Human antibodies specific to kdr and uses thereof

ABSTRACT

The invention provides an antibodies that bind to KDR with an affinity comparable to or higher than human VEGF, and that neutralizes activation of KDR. Antibodies include whole immunoglobulins, monovalent Fabs and single chain antibodies, multivalent single chain antibodies, diabodies, triabodies, and single domain antibodies. The invention further provides nucleic acids and host cells that encode and express these antibodies. The invention further provides a method of neutralizing the activation of KDR, a method of inhibiting angiogenesis in a mammal and a method of inhibiting tumor growth in a mammal.

FIELD OF THE INVENTION

The present invention is directed to human antibodies that bind to KDR,that block binding of KDR to vascular endothelial growth factor receptor(VEGFR), and that neutralize activation of KDR. The antibodies are usedfor treating neoplastic diseases and hyperproliferative disorders andcan be used alone or in combination with other VEGFR antagonists andwith epidermal growth factor receptor (EGFR) antagonists.

BACKGROUND OF THE INVENTION

Angiogenesis is a highly complex process of developing new blood vesselsthat involves the proliferation and migration of, and tissueinfiltration by capillary endothelial cells from pre-existing bloodvessels, cell assembly into tubular structures, joining of newly formingtubular assemblies to closed-circuit vascular systems, and maturation ofnewly formed capillary vessels.

Angiogenesis is important in normal physiological processes includingembryonic development, follicular growth, and wound healing, as well asin pathological conditions such as tumor growth and in non-neoplasticdiseases involving abnormal neovascularization, including neovascularglaucoma (Folkman, J. and Klagsbrun, M., Science, 235:442-7 (1987).Other disease states include but are not limited to, neoplasticdiseases, including but not limited to solid tumors, atherosclerosis andother inflammatory diseases such as rheumatoid arthritis, andophthahnological conditions such as diabetic retinopathy and age-relatedmacular degeneration. Conditions or diseases to which persistent oruncontrolled angiogenesis contribute have been termed angiogenicdependent or angiogenic associated diseases.

One means for controlling such diseases and pathological conditionscomprises restricting the blood supply to those cells involved inmediating or causing the disease or condition, for example, by occludingblood vessels supplying portions of organs in which tumors are present.Such approaches require the site of the tumor to be identified and aregenerally limited to treatment to a single site, or a small number ofsites. An additional disadvantage of direct mechanical restriction of ablood supply is that collateral blood vessels develop, often quiterapidly, restoring the blood supply to the tumor.

Other approaches have focused on the modulation of factors that areinvolved in the regulation of angiogenesis. While usually quiescent,vascular endothelial proliferation is highly regulated, even duringangiogenesis. VEGF is a factor that has been implicated as a regulatorof angiogenesis in vivo (Klagsbrun, M. and D'Amore, P., Annual Rev.Physiol., 53: 217-39 (1991)).

An endothelial-cell specific mitogen, VEGF, acts as an angiogenesisinducer by specifically promoting the proliferation of endothelialcells. It is a homodimeric glycoprotein consisting of two 23 kDsubunits. Four different monomeric isoforms of VEGF resulting fromalternative splicing of mRNA have been identified. These include twomembrane bound forms (VEGF₂₀₆ and VEGF₁₈₉) and two soluble forms(VEGF₁₆₅ and VEGF₁₂₁). VEGF₁₆₅ is the most abundant isoform in all humantissues except placenta.

VEGF is expressed in embryonic tissues (Breier et al., Development,114:521-32 (1992)), macrophages, and proliferating epidermalkeratinocytes during wound healing (Brown et al., J. Exp. Med.,176:1375-9 (1992)), and may be responsible for tissue edema associatedwith inflammation (Ferrara et al., Endocr. Rev., 13:18-32 (1992)). Insitu hybridization studies have demonstrated high levels of VEGFexpression in a number of human tumor lines including glioblastomamultiforme, hemangioblastoma, other central nervous system neoplasms andAIDS-associated Kaposi's sarcoma (Plate, K. et al., Nature, 359:845-8(1992); Plate, K. et al., Cancer Res., 53:5822-7 (1993); Berkman, R. etal., J. Clin. Invest., 91:153-9 (1993); Nakamura, S. et al., AIDSWeekly, 13 (1) (1992)). High levels of VEGF expression has also beenfound in atherosclerotic lesions, plaques and in inflammatory cells.

VEGF mediates its biological effect through high affinity VEGF receptorswhich are selectively expressed on endothelial cells during, forexample, embryogenesis (Millauer, B. et al. Cell, 72:835-46 (1993)) andtumor formation, and which have been implicated in modulatingangiogenesis and tumor growth. These receptors comprise a tyrosinekinase cytosolic domain that initiates the signaling pathway involved incell growth.

VEGF receptors typically are class III receptor-type tyrosine kinasescharacterized by having several, typically 5 or 7, immunoglobulin-likeloops in their amino-terminal extracellular receptor ligand-bindingdomains (Kaipainen et al., J. Exp. Med., 178:2077-88 (1993)). The othertwo regions include a transmembrane region and a carboxy-terminalintracellular catalytic domain interrupted by an insertion ofhydrophilic interkinase sequences of variable lengths, called the kinaseinsert domain (Terman et al., Oncogene, 6:1677-83 (1991)). VEGFreceptors include fms-like tyrosine kinase receptor (flt-1), or VEGFR-1,sequenced by Shibuya et al., Oncogene, 5:519-24 (1990), kinase insertdomain-containing receptor/fetal liver kinase (KDR/flk-1), or VEGFR-2,described in WO 92/14248, filed Feb. 20, 1992, and Terman et al.,Oncogene, 6:1677-83 (1991) and sequenced by Matthews et al., Proc. Natl.Acad. Sci. USA, 88:9026-30 (1991), although other receptors can alsobind VEGF. Another tyrosine kinase receptor, VEGFR-3 (fit-4), binds theVEGF homologues VEGF-C and VEGF-D and is important in the development oflymphatic vessels.

Release of VEGF by a tumor mass stimulates angiogenesis in adjacentendothelial cells. When VEGF is expressed by the tumor mass, endothelialcells adjacent to the VEGF+ tumor cells will up-regulate expression ofVEGF receptors, e.g., VEGFR-1 and VEGFR-2. It is generally believed thatKDR/VEGFR-2 is the main VEGF signal transducer that results inendothelial cell proliferation, migration, differentiation, tubeformation, increase of vascular permeability, and maintenance ofvascular integrity. VEGFR-1 possesses a much weaker kinase activity, andis unable to generate a mitogenic response when stimulated by VEGF,although it binds to VEGF with an affinity that is approximately 10-foldhigher than KDR. VEGFR-1 has also been implicated in VEGF and placentagrowth factor (P1GF) induced migration of monocytes and macrophages andproduction of tissue factor.

High levels of VEGFR-2, for example, are expressed by endothelial cellsthat infiltrate gliomas (Plate, K. et al. (1992)), and are specificallyupregulated by VEGF produced by human glioblastomas (Plate, K. et al.(1993)). The finding of high levels of VEGR-2 expression in glioblastomaassociated endothelial cells (GAEC) suggests that receptor activity isinduced during tumor formation, since VEGFR-2 transcripts are barelydetectable in normal brain endothelial cells, indicating generation of aparacrine VEGF/EGFR loop. This upregulation is confined to the vascularendothelial cells in close proximity to the tumor. Blocking VEGFactivity with neutralizing anti-VEGF monoclonal antibodies (mAbs)results in inhibition of the growth of human tumor xenografts in nudemice (Kim, K. et al. Nature, 362:841-4 (1993)), suggesting a direct rolefor VEGF in tumor-related angiogenesis.

Accordingly, VEGFR antagonists have been developed to treat vascularizedtumors and other angiogenic diseases. These have included neutralizingantibodies that block signaling by VEGF receptors expressed on vascularendothelial cells to reduce tumor growth by blocking angiogenesisthrough an endothelial-dependent paracrine loop. See, e.g., U.S. Pat.No. 6,365,157 (Rockwell et al.), WO 00/44777 (Zhu et al.), WO 01/54723(Kerbel); WO 01/74296 (Witte et al.), WO 01/90192 (Zhu), WO 03/002144(Zhu), and WO 03/000183 (Carmeliet et al.).

VEGF receptors have also been found on some non-endothelial cells, suchas tumor cells producing VEGF, wherein an endothelial-independentautocrine loop is generated to support tumor growth. For example, VEGFis almost invariably expressed by all established leukemic cell linesand freshly isolated human leukemias. Further, VEGFR-2 and VEGFR-1 areexpressed by certain human leukemias. Fielder et al., Blood 89:1870-5(1997); Bellamy et al., Cancer Res. 59728-33 (1999). It has beendemonstrated that a VEGF/human VEGFR-2 autocrine loop mediates leukemiccell survival and migration in vivo. Dias et al., J. Clin. Invest.106:511-21 (2000); and WO 01/74296 (Witte et al.). Similarly, VEGFproduction and VEGFR expression also have been reported for some solidtumor cell lines in vitro. (See, Sato, K. et al., Tohoku J. Exp. Med.,185: 173-84 (1998); Ishii, Y., Nippon Sanka Fujinka Gakkai Zasshi, 47:133-40 (1995); and Ferrer, F. A. et al, Urology, 54:567-72 (1999)). Ithas further been demonstrated that VEGFR-1 Mabs inhibit an autocrineVEGFR/human VEGFR-1 loop in breast carcinoma cells. Wu, et al.,“Monoclonal antibody against VEGFR1 inhibits flt1-positive DU4475 humanbreast tumor growth by a dual mechanism involving anti-angiogenic andtumor cell growth inhibitory activities,” AACR NCI EORTC InternationalConference on Molecular Targets and Cancer Therapeutics, Oct. 29-Nov. 2,2001, Abstract #7.

There remains a need for agents0 which inhibit VEGF receptor activity totreat or prevent VEGF-receptor dependent diseases or conditions, byinhibiting, for example, pathogenic angiogenesis or tumor growth throughinhibition of the paracrine and/or autocrine VEGF/VEGFR loop.

SUMMARY OF THE INVENTION

The present invention provides human antibodies, and portions thereofthat bind to KDR, block binding of vascular endothelial growth factor(VEGF) to KDR, and neutralize activation of KDR. The antibodies are usedfor treating neoplastic diseases, including, for example, solid andnon-solid tumors. The antibodies can also be used for treatment ofhyperproliferative disorders. Accordingly, the invention providesmethods of neutralizing the activation of KDR, methods of inhibitingtumor growth, including inhibition of tumor associated angiogenesis, andmethods of treating other angiogenesis related disorders. The presentinvention provides kits having human antibodies or antibody fragmentsthat bind to VEGR receptors.

The antibodies can be used alone or in combination with other VEGFRantagonists, and/or angiogenesis inhibitors such as, for example,epidermal growth factor receptor (EGFR) antagonists. The invention alsoprovides nucleic acid molecules that encode the antibodies.

Abbreviations—VEGF, vascular endothelial growth factor; bFGF, basicfibroblast growth factor; KDR, kinase insert domain-containing receptor(also known as VEGF receptor 2); FLK-1, fetal liver kinase 1; scFv,single chain Fv; HUVEC, human umbilical vein endothelial cells; PBS,0.01M phosphate buffered saline (pH 7.2); PBST, PBS containing 0.1%Tween-20; AP, alkaline phosphatase; EGF, epidermal growth factor; V_(H)and V_(L), variable domain of immunogloblin heavy and light chain,respectively.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the identification and expression of human anti-DKR Fabfragments. FIG. 1A: BstN I digestion patterns of four neutralizinganti-KDR Fab. FIG. 1B: SDS-PAGE analysis of purified Fab fragments undernonreducing conditions. Lane 1, D1F7; Lane 2, D2C6; Lane 3, D1H4; Lane4, D2H2.

FIG. 2 depicts binding to KDR, blocking of KDR/VEGF interaction andblocking of Flk-1VEGF interaction by human anti-KDR Fab fragments.

FIG. 2A: Dose-dependent binding of human anti-KDR Fab to immobilizedKDR. FIG. 2B: Inhibition of KDR binding to immobilized VEGF by anti-KDRFab. FIG. 2C: Inhibition of Flk-1 binding to immobilized VEGF byanti-KDR Fab. Various amounts of Fab proteins were incubated with afixed amount of KDR-AP (2B) or Flk-1-AP (2C) in solution at RT for 1 h.

FIG. 3 depicts epitope mapping for the anti-KDR Fab fragments. KDR-AP,its domain deletion-AP variants, and Flk-1-AP were captured on a 96-wellplate and incubated with human anti-KDR Fab fragments. Data arepresented relative to binding of the Fab fragments to full-length KDR.

FIG. 4 depicts inhibition of VEGF-induced HUVEC mitogenesis by humananti-KDR Fab fragments. Various amounts of anti-KDR Fab fragments wereadded to duplicate wells and incubated at 37° C. for 1 h, after whichVEGF was added to the wells to a final concentration of 16 ng/ml. Cellswere harvested and DNA incorporated radioactivity was determined.

FIG. 5 depicts inhibition of VEGF-stimulated migration of human leukemiacells by the anti-KDR Fab fragments. FIG. 5A: VEGF promotes migration ofHL60 and HEL cells in a dose dependent manner. FIG. 5B: Inhibition ofVEGF-stimulated migration of human leukemia cells by the anti-KDR Fabfragments. The amount of KDR-AP that bound to the immobilized VEGF wasquantified by incubation of the plates with AP substrate and reading ofA405 nm.

FIG. 6 depicts binding to KDR and blocking of KDR/VEGF interaction byhuman anti-KDR antibodies. FIG. 6A: Dose-dependent binding of anti-KDRto immobilized KDR. Various amounts of antibodies were incubated at RTfor 1 h in 96-well plates coated with KDR FIG. 6B: Inhibition of bindingof KDR to immobilized VEGF by human anti-KDR antibodies. Various amountsof the antibodies were incubated with a fixed amount of KDR-AP insolution at RT for 1 hr.

FIG. 7 depicts inhibition of VEGF binding and VEGF-induced mitogenesisof HUVEC. FIG. 7A: inhibition of binding of radiolabeled VEGF tocell-surface KDR by human anti-KDR antibodies. Various amounts ofanti-KDR antibodies were mixed with 2 ng of ¹²⁵I labeled VEGF₁₆₅ andadded to a 80-90% confluent monolayer of HUVEC cells. The cells wereincubated at RT for 2 h, washed and bound radioactivity was determined.FIG. 7B: Inhibition of VEGF-induced HUVEC mitogenesis by human anti-KDRantibodies. Various amounts of human anti-KDR antibodies were incubatedwith HUVEC cells for 1 h, followed by addition of VEGF. Cells wereharvested and DNA incorporated radioactivity was determined.

FIG. 8 depicts expression of KDR and VEGF by human leukemia cells. FIG.8A: selected miRNA levels were determined by RT-PCR. Lane 1: molecularweight markers; 1000, 850, 650, 500, 400 bp; Lane 2: negative control;Lane 3: HL60 cells promyelocytic); Lane 4: HEL cells (megakaryocytic);Lane 5: U937 cells (hisitocytic); Lane 6: HUVEC. FIG. 8B: Secretion ofVEGF by human leukemia cells cultured with 10% FCS or in serum-freemedia.

FIG. 9 depicts inhibition of VEGF-stimulated migration of human leukemiacells by human anti-KDR antibodies. FIG. 9A: HL60 cells. FIG. 9B: HELcells. FIG. 9C: U937 cells.

FIG. 10 depicts inhibition of leukemia advancement in vivo as determinedby survival rates. Sublethally irradiated NOD-SCID mice were innoculatedwith 2×10⁷ HL60 cells and treated with various doses of IMC-1C11,IMC-2C6 or IMC-1121 via intraperitoneal injection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies that bind specifically to anextracellular domain of VEGFR-2 (KDR). The antibodies comprise humanV_(H) and V_(L) framework regions (FWs) as well as human complementarydetermining regions (CDRs). Preferably, the entire V_(H) and V_(L)variable domains are human or derived from human sequences. For example,a variable domain of the invention may be obtained from a peripheralblood lymphocyte that contains a rearranged variable region gene.Alternatively, variable domain portions, such as CDR and FW regions, maybe derived from different human sequences. In another example, a humanV_(H) variable domain is encoded by a human V_(H) gene segment and asynthetic sequence for the CDR3H region (i.e., a synthetic D_(H)-J_(H)gene segment. Likewise, a human V_(L) variable domain may be encoded bya human V_(L) gene segment and a synthetic sequence for the CDR3L region(i.e., a synthetic D_(H)-J_(H) gene segment).

Antibodies of the present invention also include those for which bindingcharacteristics have been improved by direct mutation, methods ofaffinity maturation, phage display, or chain shuffling. Affinity andspecificity may be modified or improved by mutating CDRs and screeningfor antigen binding sites having the desired characteristics (see, e.g.,Yang et al., J. Mol. Biol., 254: 392-403 (1995)). CDRs are mutated in avariety of ways. One way is to randomize individual residues orcombinations of residues so that in a population of otherwise identicalantigen binding sites, all twenty amino acids are found at particularpositions. Alternatively, mutations are induced over a range of CDRresidues by error prone PCR methods (see, e.g., Hawkins et al., J. Mol.Biol., 226: 889-896 (1992)). For example, phage display vectorscontaining heavy and light chain variable region genes may be propagatedin mutator strains of E. coli (see, e.g., Low et al., J. Mol. Biol.,250: 359-368 (1996)). These methods of mutagenesis are illustrative ofthe many methods known to one of skill in the art.

The antibodies bind to KDR and neutralize activation, for example, byblocking receptor dimerization and/or VEGF binding. Antibodies of theinvention can be used to neutralize VEGFR activation in vitro or invivo. by binding to an extracellular domain of a VEGF receptor.Extracellular domains of a VEGF receptor include, for example, aligand-binding domain on an extracellular portion of the receptor. Invivo, the antibodies inhibit angiogenesis, and/or reduce tumor growth,

Antibodies are proteins that recognize and bind to a specific antigen orsubstance. The antibodies of the present invention bind KDR at least asstrongly as the natural ligand. Affinity, represented by the equilibriumconstant for the dissociation of an antigen with an antibody (Kd),measures the binding strength between an antigenic determinant and anantibody binding site. Avidity is the measure of the strength of bindingbetween an antibody with its antigen. Avidity is related to both theaffinity between an epitope with its antigen binding site on theantibody, and the valence of the antibody. Valency refers to the numberof antigen binding sites which an immunoglobulin has for a particularepitope. For example, a monovalent antibody has one binding site for aparticular epitope. An antigenic determinant, or epitope, is the site onan antigen at which a given antibody binds. Typical values of K are 10⁵to 10¹¹ liters/mol. Any K less than 10⁴ liters/mol is considered toindicate binding which is nonspecific. The reciprocal of K is designatedas K_(d). (K_(d) also may be referred to as the dissociation constant.)The lesser the value of the K_(d), the stronger the binding strengthbetween an antigenic determinant and the antibody binding site.

The natural ligand of KDR is human VEGF. VEGF binds KDR with an affinity(K_(d)) of about 0.93 nM. In order to hinder the binding of VEGF withKDR, an anti-KDR antibody should bind KDR at lease as strongly as VEGF.In other words, the anti-KDR antibody needs to successfully compete withVEGF with respect to binding KDR. An antibody with a K_(d) of at most 5nM is considered to bind as strongly as the natural ligand. Theantibodies of the invention preferably bind KDR with an affinity of atmost about 4 nM, more preferably with an affinity of at most about 3 nM,most preferably with an affinity of at most about 2 nM, and optimallywith an affinity of at most about 1 nM. The avidity of bivalentantibodies will, of course, be greater than the affinity. Bivalentantibodies preferably bind KDR with an avidity greater than 0.5 nM, morepreferably greater than 0.25 nM, and optimally greater than 0.1 nM.

The antibodies of the invention neutralize KDR. (See Examples.) In thisspecification, neutralizing a receptor means diminishing and/orinactivating the intrinsic kinase activity of the receptor to transducea signal. A reliable assay for KDR neutralization is the inhibition ofreceptor phosphorylation.

The present invention is not limited by any particular mechanism of KDRneutralization. The mechanism followed by one antibody is notnecessarily the same as that followed by another. Some possiblemechanisms include preventing binding of the VEGF ligand to theextracellular binding domain of the KDR, and preventing dimerization oroligomerization of receptors. Other mechanisms cannot, however, be ruledout.

Antibodies of the invention include, but are not limited to, naturallyoccurring antibodies, bivalent fragments such as (Fab′)₂, monovalentfragments such as Fab, single chain antibodies, single chain Fv (scFv),single domain antibodies, multivalent single chain antibodies,diabodies, triabodies, and the like that bind specifically withantigens.

Monovalent single chain antibodies (i.e., scFv) include an antibodyvariable heavy-chain fragment (V_(H)) linked to an antibody variablelight-chain fragment (V_(L)) by a peptide linker which allows the twofragments to associate to form a functional antigen binding site (see,for example U.S. Pat. No. 4,946,778 (Ladner et al.), WO 88/09344,(Huston et al.). WO 92/01047 (McCafferty et al.) describes the displayof scFv fragments on the surface of soluble recombinant genetic displaypackages, such as bacteriophage. A single chain antibody with a linker(L) can be represented as V_(L)-L-V_(H) or V_(H)-L-V_(L).

Each domain of the antibodies of this invention may be a completeantibody heavy or light chain variable domain, or it may be a functionalequivalent or a mutant or derivative of a naturally occurring domain, ora synthetic domain constructed, for example, in vitro using a techniquesuch as one described in WO 93/11236 (Griffiths et al.). For instance,it is possible to join together domains corresponding to antibodyvariable domains which are missing at least one amino acid. Theimportant characterizing feature is the ability of each domain toassociate with a complementary domain to form an antigen binding site.Accordingly, the terms “variable heavy/light chain fragment” should notbe construed to exclude variants which do not have a material effect onhow the invention works.

Functional equivalents of the invention include polypeptides with aminoacid sequences substantially the same as the amino acid sequence of thevariable or hypervariable regions of the full length KDR antibodies.“Substantially the same” amino acid sequence is defined herein as asequence with at least 70%, preferably at least about 80%, and morepreferably at least about 90% homology to another amino acid sequence,as determined by the FASTA search method in accordance with Pearson andLipman, Proc. Natl. Acad. Sci. USA 85, 2444-8 (1988).

Single domain antibodies have a single variable domain that is capableof efficiently binding antigen. Examples of antibodies wherein bindingaffinity and specificity are contributed primarily by one or the othervariable domain are known in the art. See, e.g., Jeffrey, P. D. et al.,Proc. Natl. Acad. Sci. USA 90:10310-4 (1993), which discloses ananti-digoxin antibody which binds to digoxin primarily by the antibodyheavy chain. Accordingly, single antibody domains can be identified thatbind well to VEGF receptors. Such antibody domains can be obtained, forexample, from naturally occurring antibodies, or Fab or scFv phagedisplay libraries. It is understood that, to make a single domainantibody from an antibody comprising a V_(H) and a V_(L) domain, certainamino acid substitutions outside the CDR regions may be desired toenhance binding, expression or solubility. For example, it may bedesirable to modify amino acid residues that would otherwise be buriedin the V_(H)-V_(L) interface.

More recently, antibodies that are homodimers of heavy chains have beendiscovered in camelids (camels, dromedaries and llamas). These heavychain antibodies are devoid of light chains and the first constantdomain. (See, e.g., Muyldermans, S., 2001, J. Biotechnol. 74:277-302)The reduced-size antigen binding fragments are well expressed inbacteria, bind to antigen with high affinity, and are very stable. Phagedisplay libraries of single domain antibodies (i.e., having a singlevariable domain that can be a light chain or a heavy chain variabledomain) can be produced and screened in the same manner as scFv and Fablibraries. Scaffolds for such single domain antibodies can be modifiedmouse or human variable domains. It is noted that single antibodydomains can bind antigen in a variety of antigen binding modes. That is,the primary antibody-antigen interactions are not limited to amino acidresidues corresponding to CDRs of V_(H)-V_(L) containing antibodies, andconsideration can be given to binding interactions outside of CDRresidues when optimizing the binding characteristics of such antibodies.

Single chain antibodies lack some or all of the constant domains of thewhole antibodies from which they are derived. Therefore, they mayovercome some of the problems associated with the use of wholeantibodies. For example, single-chain antibodies tend to be free ofcertain undesired interactions between heavy-chain constant regions andother biological molecules. Additionally, single-chain antibodies areconsiderably smaller than whole antibodies and may have greaterpermeability than whole antibodies, allowing single-chain antibodies tolocalize and bind to target antigen-binding sites more efficiently.Also, single chain antibodies can be produced on a relatively largescale in prokaryotic cells, thus facilitating their production.Furthermore, the relatively small size of single-chain antibodies makesthem less likely to provoke an unwanted immune response in a recipientthan whole antibodies.

The peptide linkers used to produce the single chain antibodies may beflexible peptides selected to assure that the proper three-dimensionalfolding of the V_(L) and V_(H) domains may occur once they are linked soas to maintain the target molecule binding-specificity of the fulllength anti-KDR antibody. Generally, the carboxyl terminus of the V_(L)or V_(H) sequence may be covalently linked by such a peptide linker tothe amino acid terminus of a complementary V_(H) or V_(L) sequence. Thelinker is generally 10 to 50 amino acid residues. Preferably, the linkeris 10 to 30 amino acid residues. More preferably the linker is 12 to 30amino acid residues. Most preferably is a linker of 15 to 25 amino acidresidues. An example of such linker peptides include(Gly-Gly-Gly-Gly-Ser)₃.

Single chain antibodies, each having one V_(H) and one V_(L) domaincovalently linked by a first peptide linker, can be covalently linked byat least one more peptide linker to form a multivalent single chainantibody. Multivalent single chain antibodies allow for the constructionof antibody fragments which have the specificity and avidity of wholeantibodies, but lack the constant regions of the full length antibodies.

Multivalent antibodies may be monospecific or multispecific. The termspecificity refers to the number of different types of antigenicdeterminants to which a particular antibody can bind. If the antibodybinds to only one type of antigenic determinant, the antibody ismonospecific. If the antibody binds to different types of antigenicdeterminants then the antibody is multispecific.

For example, a bispecific multivalent single chain antibody allows forthe recognition of two different types of epitopes. The epitopes mayboth be on KDR. Alternatively, one epitope may be on KDR, and the otherepitope may be on another antigen.

Each chain of a multivalent single chain antibody includes a variablelight-chain fragment and a variable heavy-chain fragment, and is linkedby a peptide linker to at least one other chain. The peptide linker iscomposed of at least fifteen amino acid residues. The maximum number ofamino acid residues is about one hundred. In a preferred embodiment, thenumber of V_(L) and V_(H) domains is equivalent. Preferably, the peptidelinker (L₁) joining the V_(H) and V_(L) domains to form a chain and thepeptide linker (L₂) joining two or more chains to form a multivalentscFv have substantially the same amino acid sequence.

For example, a bivalent single chain antibody can be represented asfollows: V_(L)-L₁-V_(H)-L₂-V_(L)-L₁-V_(H); orV_(L)-L₁-V_(H)-L₂-V_(H)-L₁-V_(H); or V_(H)-L₁-V_(L)-L₂-V_(H)-L₁-V_(L);or V_(H)-L₁-V_(L)-L₂-V_(L)-L₁-V_(H).

Multivalent single chain antibodies which are trivalent or greater haveone or more antibody fragments joined to a bivalent single chainantibody by additional peptide linkers. One example of a trivalentsingle chain antibody is:V_(L)-L₁-V_(H)-L₂-V_(L)-L₁-V_(H)-L₂-V_(L)-L₁-V_(H).

Two single chain antibodies can be combined to form a diabody, alsoknown as a bivalent dimer. Diabodies have two chains and two bindingsites, and may be monospecific or bispecific. Each chain of the diabodyincludes a V_(H) domain connected to a V_(L) domain. The domains areconnected with linkers that are short enough to prevent pairing betweendomains on the same chain, thus driving the pairing betweencomplementary domains on different chains to recreate the twoantigen-binding sites. Accordingly, one chain of a bispecific diabodycomprises V_(H) of a first specificity and V_(L) of a secondspecificity, whereas the second chain comprises V_(H) of the secondspecificity and V_(L) of the first specificity. The peptide linkerincludes at least five amino acid residues and no more than ten aminoacid residues, e.g. (Gly-Gly-Gly-Gly-Ser), (Gly-Gly-Gly-Gly-Ser)₂. (SEQID NO:19.) The diabody structure is rigid and compact. Theantigen-binding sites are at opposite ends of the molecule.

Three single chain antibodies can be combined to form triabodies, alsoknown as trivalent trimers. Triabodies are constructed with the aminoacid terminus of a V_(L) or V_(H) domain directly fused to the carboxylterminus of a V_(L) or V_(H) domain, i.e., without any linker sequence.The triabody has three Fv heads with the polypeptides arranged in acyclic, head-to-tail fashion. A possible conformation of the triabody isplanar with the three binding sites located in a plane at an angle of120 degrees from one another. Triabodies may be monospecific, bispecificor trispecific.

Preferably the antibodies of this invention contain all sixcomplementarity determining regions of the whole antibody, althoughantibodies containing fewer than all of such regions, such as three,four or five CDRs, are also functional.

To minimize the immunogenicity of antibodies that bind to VEGFreceptors, the present invention provides antibodies which comprisehuman variable and constant domain sequences. The antibodies are derivedfrom a human source and bind to an extacellular domain of KDR andneutralize activation of the receptor. DNA encoding human antibodies maybe prepared by recombining DNA encoding human constant regions and DNAencoding variable regions derived from humans. For example, antibodiesof the invention can be obtained by screening libraries consisting ofcombinations of human light chain and heavy chain variable domains. Thenucleic acids from which the antibodies are expressed can be somaticallymutated, or be germline sequences derived from naive B cells.

DNA encoding human antibodies may be prepared by recombining DNAencoding human constant regions and variable regions, other than theCDRs, derived substantially or exclusively from the corresponding humanantibody regions and DNA encoding CDRs derived from a human.

Suitable sources of DNAs that encode fragments of antibodies include anycell, such as hybridomas and spleen cells, that express the full lengthantibody. Another source is single chain antibodies produced from aphage display library as is known in the art.

The antibodies of this invention may be or may combine members of anyimmunoglobulin class, such as IgG, IgM, IgA, IgD, or IgE, and thesubclasses thereof.

The protein used to identify VEFGR binding antibodies of the inventionis usually KDR, and is normally limited to the extracellular domain ofKDR. The KDR extracellular domain may be free or conjugated to anothermolecule.

In the examples below high affinity anti-KDR antibodies, which blockVEGF binding to KDR, were isolated from a phage display libraryconstructed from human heavy chain and light chain variable regiongenes. Over 90% of recovered clones after three rounds of selection arespecific to KDR. The binding affinities for KDR of the screened Fabs arein the nM range, which are as high as those of several bivalent anti-KDRmonoclonal antibodies produced using hybridoma technology.

The antibodies of this invention may be fused to additional amino acidresidues. Such residues may be a peptide tag, perhaps to facilitateisolation, or they may be a signal sequence for secretion of thepolypeptide from a host cell upon synthesis. Suitably, secretory leaderpeptides are used, being amino acids joined to the N-terminal end of apolypeptide to direct movement of the polypeptide out of the cytosol.

The present invention also provides nucleic acids which comprise asequence encoding a polypeptide according to the invention, and diverserepertoires of such nucleic acid.

Antibodies of the invention neutralize activation of KDR. One measure ofKDR neutralization is inhibition of the tyrosine kinase activity of thereceptor. Tyrosine kinase inhibition can be determined using well-knownmethods. The antibodies of the present invention generally causeinhibition or regulation of phosphorylation events. Accordingly,phosphorylation assays are useful in determining antibodies useful inthe context of the present invention. Tyrosine kinase inhibition may bedetermined by measuring the autophosphorylation level of recombinantkinase receptor, and/or phosphorylation of natural or syntheticsubstrates. Phosphorylation can be detected, for example, using anantibody specific for phosphotyrosine in an ELISA assay or on a westernblot. Some assays for tyrosine kinase activity are described in Panek etal., J. Pharmacol. Exp. Thera., 283: 1433-44 (1997) and Batley et al.,Life Sci., 62: 143-50 (1998).

In addition, methods for detection of protein expression can beutilized, wherein the proteins being measured are regulated by KDRtyrosine kinase activity. These methods include immunohistochemistry(IHC) for detection of protein expression, fluorescence in situhybridization (FISH) for detection of gene amplification, competitiveradioligand binding assays, solid matrix blotting techniques, such asNorthern and Southern blots, reverse transcriptase polymerase chainreaction (RT-PCR) and ELISA. See, e.g., Grandis et al., Cancer,78:1284-92. (1996); Shimizu et al., Japan J. Cancer Res., 85:567-71(1994); Sauter et al., Am. J. Path., 148:1047-53 (1996); Collins, Glia,15:289-96 (1995); Radinsky et al., Clin. Cancer Res., 1:19-31 (1995);Petrides et al., Cancer Res., 50:3934-39 (1990); Hoffiann et al.,Anticancer Res., 17:4419-26 (1997); Wikstrand et al., Cancer Res.,55:3140-48 (1995).

In vivo assays can also be utilized. For example, receptor tyrosinekinase inhibition can be observed by mitogenic assays using cell linesstimulated with receptor ligand in the presence and absence ofinhibitor. For example, HUVEC cells (ATCC) stimulated with VEGF can beused to assay VEGFR inhibition. Another method involves testing forinhibition of growth of VEGF-expressing tumor cells, using for example,human tumor cells injected into a mouse. See, U.S. Pat. No. 6,365,157(Rockwell et al.).

In the methods of the present invention, a therapeutically effectiveamount of an antibody of the invention is administered to a mammal inneed thereof. The term “administering” as used herein means deliveringthe antibodies of the present invention to a mammal by any method thatmay achieve the result sought. They may be administered, for example,intravenously or intramuscularly. Although human antibodies of theinvention are particularly useful for administration to humans, they maybe administered to other mammals as well. The term “mammal” as usedherein is intended to include, but is not limited to, humans, laboratoryanimals, domestic pets and farm animals. “Therapeutically effectiveamount” means an amount of antibody of the present invention that, whenadministered to a mammal, is effective in producing the desiredtherapeutic effect, such as inhibiting kinase activity.

While not intended to be bound to any particular mechanism, the diseasesand conditions which may be treated or prevented by the present methodsinclude, for example, those in which pathogenic angiogenesis or tumorgrowth is stimulated through a VEGFR paracrine and/or autocrine loop.

Neutralization of activation of a VEGF receptor in endothelial ornon-endothelial cells, such as tumor cells, may be performed in vitro orin vivo. Neutralizing VEGF activation of a VEGF receptor in a sample ofVEGF-receptor expressing cells comprises contacting the cells with anantagonist, e.g., an antibody, of the invention. The cells are contactedin vitro with the antagonist, e.g., the antibody, before, simultaneouslywith, or after, adding VEGF to the cell sample.

In vivo, an antibody of the invention is contacted with a VEGF receptorby administration to a mammal, preferably a human. An in vivoneutralization method is useful for inhibiting tumor growth,angiogenesis associated with tumor growth, or other pathologic conditionassociated with angiogenesis, in a mammal. Accordingly, the antibodiesof the invention are anti-angiogenic and anti-tumor immunotherapeuticagents.

Tumors which may be treated include primary tumors and metastatictumors, as well as refractory tumors. Refractory tumors include tumorsthat fail to respond or are resistant to treatment with chemotherapeuticagents alone, antibodies alone, radiation alone or combinations thereof.Refractory tumors also encompass tumors that appear to be inhibited bytreatment with such agents, but recur up to five years, sometimes up toten years or longer after treatment is discontinued.

Antibodies of the present invention are useful for treating tumors thatexpress VEGF receptors, especially KDR. Such tumors arecharacteristically sensitive to VEGF present in their environment, andmay further produce and be stimulated by VEGF in an autocrinestimulatory loop. The method is therefore effective for treating a solidor non-solid tumor that is not vascularized, or is not yet substantiallyvascularized. Examples of solid tumors which may be accordingly treatedinclude breast carcinoma, lung carcinoma, colorectal carcinoma,pancreatic carcinoma, glioma and lymphoma. Some examples of such tumorsinclude epidermoid tumors, squamous tumors, such as head and necktumors, colorectal tumors, prostate tumors, breast tumors, lung tumors,including small cell and non-small cell lung tumors, pancreatic tumors,thyroid tumors, ovarian tumors, and liver tumors. Other examples includeKaposi's sarcoma, CNS neoplasms, neuroblastomas, capillaryhemangioblastomas, meningiomas and cerebral metastases, melanoma,gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma,glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.Examples of vascularized skin cancers for which the antagonists of thisinvention are effective include squamous cell carcinoma, basal cellcarcinoma and skin cancers that can be treated by suppressing the growthof malignant keratinocytes, such as human malignant keratinocytes.

Examples of non-solid tumors include leukemia, multiple myeloma andlymphoma. Some examples of leukemias include acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia(ALL), chronic lymphocytic leukemia (CLL), erythrocytic leukemia ormonocytic leukemia. Some examples of lymphomas include Hodgkin's andnon-Hodgkin's lymphoma.

Experimental results described below demonstrate that antibodies of theinvention specifically block VEGF induced stimulation of KDR (VEGFR-2)in leukemia cells. In vivo studies also described below show that theantibodies were able to significantly inhibit tumor growth in nude mice.

A cocktail of VEGF receptor antagonists, e.g., monoclonal antibodies,provides an especially efficient treatment for inhibiting the growth oftumor cells. The cocktail may include non-antibody VEGFR antagonists andmay have as few as 2, 3 or 4 receptor antagonists, and as many as 6, 8or 10.

In another aspect of the invention, anti-KDR antibodies are used toinhibit angiogenesis. VEGFR stimulation of vascular endothelium isassociated with angiogenic diseases and vascularization of tumors.Typically, vascular endothelium is stimulated in a paracrine fashion byVEGF from other sources (e.g., tumor cells).

Accordingly, the human anti-KDR antibodies are effective for treatingsubjects with vascularized tumors or neoplasms or angiogenic diseases.Such tumors and neoplasms include, for example, malignant tumors andneoplasms, such as blastomas, carcinomas or sarcomas, and highlyvascular tumors and neoplasms. Cancers that may be treated by themethods of the present invention include, for example, cancers of thebrain, genitourinary tract, lymphatic system, stomach, renal, colon,larynx and lung and bone. Non-limiting examples further includeepidermoid tumors, squamous tumors, such as head and neck tumors,colorectal tumors, prostate tumors, breast tumors, lung tumors,including lung adenocarcinoma and small cell and non-small cell lungtumors, pancreatic tumors, thyroid tumors, ovarian tumors, and livertumors. The method is also used for treatment of vascularized skincancers, including squamous cell carcinoma, basal cell carcinoma, andskin cancers that can be treated by suppressing the growth of malignantkeratinocytes, such as human malignant keratinocytes. Other cancers thatcan be treated include Kaposi's sarcoma, CNS neoplasms (neuroblastomas,capillary hemangioblastomas, meningiomas and cerebral metastases),melanoma, gastrointestinal and renal carcinomas and sarcomas,rhabdomyosarcoma, glioblastoma, including glioblastoma multiforme, andleiomyosarcoma.

A further aspect of the present invention includes methods of treatingor preventing pathologic conditions characterized by excessiveangiogenesis, involving, for example, vascularization and/orinflammation, such as atherosclerosis, rheumatoid arthritis (RA),neovascular glaucoma, proliferative retinopathy including proliferativediabetic retinopathy, macular degeneration, hemangiomas, angiofibromas,and psoriasis. Other non-limiting examples of non-neoplastic angiogenicdisease are retinopathy of prematurity (retrolental fibroplastic),corneal graft rejection, insulin-dependent diabetes mellitus, multiplesclerosis, myasthenia gravis, Chron's disease, autoimmune nephritis,primary biliary cirrhosis, acute pancreatitis, allograph rejection,allergic inflammation, contact dermatitis and delayed hypersensitivityreactions, inflammatory bowel disease, septic shock, osteoporosis,osteoarthritis, cognition defects induced by neuronal inflammation,Osler-Weber syndrome, restinosis, and fungal, parasitic and viralinfections, including cytomegaloviral infections.

The identification of such disease is well within the ability andknowledge of one skilled in the art. For example, human individuals whoare either suffering from a clinically significant neoplastic orangiogenic disease or who are at risk of developing clinicallysignificant symptoms are suitable for administration of the present VEGFreceptor antibodies. A clinician skilled in the art can readilydetermine, for example, by the use of clinical tests, physicalexamination and medical/family history, if an individual is a candidatefor such treatment.

Moreover, included within the scope of the present invention is use ofthe present antibodies in vivo and in vitro for investigative ordiagnostic methods, which are well known in the art.

The present anti-KDR antibodies can be administered for therapeutictreatments to a patient suffering from a tumor or angiogenesisassociated pathologic condition in an amount sufficient to prevent,inhibit, or reduce the progression of the tumor or pathologic condition.Progression includes, e.g, the growth, invasiveness, metastases and/orrecurrence of the tumor or pathologic condition. An amount adequate toaccomplish this is defined as a therapeutically effective dose. Amountseffective for this use will depend upon the severity of the disease andthe general state of the patient's own immune system. Dosing scheduleswill also vary with the disease state and status of the patient, andwill typically range from a single bolus dosage or continuous infusionto multiple administrations per day (e.g., every 4-6 hours), or asindicated by the treating physician and the patient's condition. Itshould be noted, however, that the present invention is not limited toany particular dose.

In an embodiment of the invention, anti-KDR antibodies can beadministered in combination with one or more other antineoplasticagents. For examples of combination therapies, see, e.g., U.S. Pat. No.6,217,866 (Schlessinger et al.) (Anti-EGFR antibodies in combinationwith antineoplastic agents); WO 99/60023 (Waksal et al.) (Anti-EGFRantibodies in combination with radiation). Any suitable antineoplasticagent can be used, such as a chemotherapeutic agent or radiation.Examples of chemotherapeutic agents include, but are not limited to,cisplatin, doxorubicin, paclitaxel, irinotecan (CPT-11), topotecan or acombination thereof. When the antineoplastic agent is radiation, thesource of the radiation can be either external (external beam radiationtherapy—EBRT) or internal (brachytherapy—BT) to the patient beingtreated. The dose of antineoplastic agent administered depends onnumerous factors, including, for example, the type of agent, the typeand severity tumor being treated and the route of administration of theagent. It should be emphasized, however, that the present invention isnot limited to any particular dose.

Further, anti-KDR antibodies of the invention may be administered withantibodies that neutralize other receptors involved in tumor growth orangiogenesis. One example of such a receptor is the VEGFR-1/Flt-1receptor. In an embodiment of the invention, an anti-KDR antibody isused in combination with a receptor antagonist that binds specificallyto VEGFR-1. Particularly preferred are antigen-binding proteins thatbind to the extracellular domain of VEGFR-1 and block binding by one orboth of its ligands, VEGF and P1GF, and/or neutralize VEGF-induced orP1GF-induced activation of VEGFR-1. For example, mAb 6.12 is a scFv thatbinds to soluble and cell surface-expressed VEGFR-1. ScFv 6.12 comprisesthe V_(L) and V_(H) domains of mouse monoclonal antibody mAb 6.12. Ahybridoma cell line producing mAb 6.12 has been deposited as ATCC numberPTA-3344 under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the regulations thereunder (BudapestTreaty).

Another example of such a receptor is EGFR. In an embodiment of thepresent invention, an anti-KDR antibody is used in combination with anEGFR antagonist. An EGFR antagonist can be an antibody that binds toEGFR or a ligand of EGFR and inhibits binding of EGFR to its ligand.Ligands for EGFR include, for example, EGF, TGF-α amphiregulin,heparin-binding EGF (HB-EGF) and betarecullulin. EGF and TGF-α arethought to be the main endogenous ligands that result in EGFR-mediatedstimulation, although TGF-α has been shown to be more potent inpromoting angiogenesis. It should be appreciated that the EGFRantagonist can bind externally to the extracellular portion of EGFR,which may or may not inhibit binding of the ligand, or internally to thetyrosine kinase domain. Examples of EGFR antagonists that bind EGFRinclude, without limitation, biological molecules, such as antibodies(and functional equivalents thereof) specific for EGFR, and smallmolecules, such as synthetic kinase inhibitors that act directly on thecytoplasmic domain of EGFR.

Other examples of growth factor receptors involved in tumorigenesis arethe receptors for platelet-derived growth factor (PDGFR), insulin-likegrowth factor (IGFR), nerve growth factor (NGFR), and fibroblast growthfactor (FGFR).

In an additional alternative embodiment, the VEGFR antagonist can beadministered in combination with one or more suitable adjuvants, suchas, for example, cytokines (IL-10 and IL-13, for example) or otherimmune stimulators. See, e.g., Larrivee et al., supra. It should beappreciated, however, that administration of only an anti-KDR antibodyis sufficient to prevent, inhibit, or reduce the progression of thetumor in a therapeutically effective manner.

In a combination therapy, the anti-KDR antibody is administered before,during, or after commencing therapy with another agent, as well as anycombination thereof, i.e., before and during, before and after, duringand after, or before, during and after commencing the antineoplasticagent therapy. For example, the anti-KDR antibody may be administeredbetween 1 and 30 days, preferably 3 and 20 days, more preferably between5 and 12 days before commencing radiation therapy.

In the present invention, any suitable method or route can be used toadminister anti-KDR antibodies of the invention, and optionally, tocoadminister antineoplastic agents and/or antagonists of otherreceptors. Routes of administration include, for example, oral,intravenous, intraperitoneal, subcutaneous, or intramuscularadministration. The dose of antagonist administered depends on numerousfactors, including, for example, the type of antagonists, the type andseverity tumor being treated and the route of administration of theantagonists. It should be emphasized, however, that the presentinvention is not limited to any particular method or route ofadministration.

It is noted that an anti-KDR antibody of the invention can beadministered as a conjugate, which binds specifically to the receptorand delivers a toxic, lethal payload following ligand-toxininternalization.

It is understood that the anti-KDR antibodies of the invention, whereused in a mammal for the purpose of prophylaxis or treatment, will beadministered in the form of a composition additionally comprising apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the binding proteins. The compositions of theinjection may, as is well known in the art, be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the mammal.

The present invention also includes kits for inhibiting tumor growthand/or angiogenesis comprising a therapeutically effective amount of ahuman anti-KDR antibody. The kits can further contain any suitableantagonist of, for example, another growth factor receptor involved intumorigenesis or angiogenesis (e.g., VEGFR-1/Flt-1, EGFR, PDGFR, IGFR,NGFR, FGFR, etc, as described above). Alternatively, or in addition, thekits of the present invention can further comprise an antineoplasticagent. Examples of suitable antineoplastic agents in the context of thepresent invention have been described herein. The kits of the presentinvention can further comprise an adjuvant, examples have also beendescribed above.

In another aspect of the invention, an anti-KDR antibody of theinvention can be chemically or biosynthetically linked to one or moreantineoplastic or antiangiogenic agents.

The invention further contemplates anti-KDR antibodies to which targetor reporter moieties are linked. Target moieties are first members ofbinding pairs. Antineoplastic agents, for example, are conjugated tosecond members of such pairs and are thereby directed to the site wherethe anti-KDR antibody is bound. A common example of such a binding pairis adivin and biotin. In a preferred embodiment, biotin is conjugated toan anti-KDR antibody, and thereby provides a target for anantineoplastic agent or other moiety which is conjugated to avidin orstreptavidin. Alternatively, biotin or another such moiety is linked toan anti-KDR antibody of the invention and used as a reporter, forexample in a diagnostic system where a detectable signal-producing agentis conjugated to avidin or streptavidin.

Accordingly, the present receptor antagonists thus can be used in vivoand in vitro for investigative, diagnostic, prophylactic, or treatmentmethods, which are well known in the art. Of course, it is to beunderstood and expected that variations in the principles of inventionherein disclosed can be made by one skilled in the art and it isintended that such modifications are to be included within the scope ofthe present invention.

All references mentioned herein are incorporated in their entirety.

EXAMPLES

The Examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be construed to, limitits scope in any way. The Examples do not include detailed descriptionsof conventional methods, such as those employed in the construction ofvectors and plasmids, the insertion of genes encoding polypeptides intosuch vectors and plasmids, or the introduction of plasmids into hostcells. Such methods are well known to those of ordinary skill in the artand are described in numerous publications including Sambrook, J., andRussell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory Press.

Example I Production of Human Fab Example I(a) Proteins and Cell Lines

Primary-cultured HUVEC were obtained from Dr. S. Rafii at CornellMedical Center, N.Y., and maintained in EBM-2 medium (Clonetics,Walkersville, Md.) at 37° C., 5% CO₂. The soluble fusion proteins,KDR-alkaline phosphatase (AP), its immunoglobulin (Ig) domain-deletionvariants, and Flk-1-AP, were expressed in stably transfected NIH 3T3 andpurified from cell culture supernatants by affinity chromatography usingimmobilized monoclonal antibody to AP as described by Lu et al., J.Biol. Chem. 275: 14321-30 (2000). VEGF₆₅ protein was expressed inbaculovirus and purified following the procedures described in Zhu etal., Cancer Res. 58: 3209-14 (1998). The leukemia cell lines, HL60 andHEL, were maintained in RPMI containing 10% fetal calf serum.

Example 1(b) Phage ELISA

Individual TG1 clones were picked and grown at 37° C. in 96 well platesand rescued with M13K07 helper phage as described above. The amplifiedphage preparation was blocked with ⅙ volume of 18% milk/PBS at RT for 1h and added to Maxi-sorp 96-well microtiter plates (Nunc) coated withKDR-AP or AP (1 μg/ml×100 μl). After incubation at RT for 1 h the plateswere washed 3 times with PBST and incubated with a rabbit anti-M13phage-HRP conjugate (Amersharn Pharmacia Biotech, Piscataway, N.J.). Theplates were washed 5 times, TMB peroxidase substrate (KPL, Gaithersburg,Md.) added, and the absorbance at 450 nm read using a microplate reader(Molecular Devices, Sunnyvale, Calif.).

Example I(c) DNA BstN I Pattern Analysis and Nucleotide Sequencing

The diversity of the anti-KDR Fab clones after each round of selectionwas analyzed by restriction enzyme digestion patterns (i.e., DNAfingerprints). The Fab gene insert of individual clones was PCRamplified using primers: PUC19 reverse, 5′ AGCGGATAACAATTTCACACAGG 3′;and fdtet seq, 5′ GTCGTCTTTCCAGACGTTAGT 3′. The amplified product wasdigested with a frequent-cutting enzyme, BstN I, and analyzed on a 3%agarose gel. DNA sequences of representative clones from each digestionpattern were determined by dideoxynucleotide sequencing.

Example I(d) Expression and Purification of Soluble Fab Fragments

Plasmids of individual clones were used to transform a nonsuppressor E.coli host HB2151. Expression of the Fab fragments in HB2151 was inducedby culturing the cells in 2YTA medium containing 1 mMisopropyl-1-thio-β-D-galactopyranoside (IPTG, Sigma) at 30° C. Aperiplasmic extract of the cells was prepared by resuspending the cellpellet in 25 mM Tris (pH 7.5) containing 20% (w/v) sucrose, 200 mM NaCl,1 mM EDTA and 0.1 mM PMSF, followed by incubation at 4° C. with gentleshaking for 1 h. After centrifugation at 15,000 rpm for 15 min, thesoluble Fab protein was purified from the supernatant by affinitychromatography using a Protein G column followed the manufacturer'sprotocol (Amersham Pharmacia Biotech).

Example I(e) Selection of Human Anti-KDR Fab from Phage Display Library

A large human Fab phage display library containing 3.7×10¹⁰ clonesDeHaard et al., J. Biol. Chem. 274: 18218-30 (1999)) was used for theselection. The library consists of PCR-amplified antibody variable lightchain genes and variable heavy chain genes fused to human constant lightchain genes (κ and λ) and DNA encoding the IgG1 heavy chain C_(H)1domain, respectively. Both heavy and light chain constructs are precededby a signal sequence—pelB for the light chain and gene III signalsequence for the heavy chain. Heavy chain constructs further encode aportion of the gene III protein for phage display, a hexahistidine tag,and an 11 amino-acid-long c-myc tag, followed by an amber codon (TAG).The hexahistidine and c-myc tags can be used for purification ordetection. The amber codon allows for phage display using suppressorhosts (such as TG1 cells) or production of Fab fragments in soluble formwhen transformed into a nonsupressor host (such as HB2151 cells).

The library stock was grown to log phase, rescued with M13-KO7 helperphage and amplified overnight in 2YTAK medium (2YT containing 100 μg/mlof ampicillin and 50 μg/ml of kanamycin) at 30° C. The phage preparationwas precipitated in 4% PEG/0.5M NaCl, resuspended in 3% fat-freemilk/PBS containing 500 μg/ml of AP protein and incubated at 37° C. for1 h to capture phage displaying anti-AP Fab fragments and to block othernonspecific binding.

KDR-AP (10 μg/ml in PBS) coated Maxisorp Star tubes (Nunc, Rosklide,Denmark) were first blocked with 3% milk/PBS at 37° C. for 1 h, and thenincubated with the phage preparation at RT for 1 h. The tubes werewashed 10 times with PBST (PBS containing 0.1% Tween-20) followed by 10times with PBS. Bound phage were eluted at RT for 10 min with 1 ml of afreshly prepared solution of 100 mM triethylamine (Sigma, St. Louis,Mo.). The eluted phage were incubated with 10 ml of mid-log phase TG1cells at 37° C. for 30 min stationary and 30 min shaking. The infectedTG1 cells were pelleted and plated onto several large 2YTAG plates andincubated overnight at 30° C. All the colonies grown on the plates werescraped into 3 to 5 ml of 2YTA medium, mixed with glycerol (10% finalconcentration), aliquoted and stored at −70° C. For the next roundselection, 100 μl of the phage stock was added to 25 ml of 2YTAG mediumand grown to mid-log phase. The culture was rescued with M13K07 helperphage, amplified, precipitated, and used for selection followed theprocedure described above, with reduced concentrations of KDR-APimmobilized on the immunotube and increased number of washes after thebinding process.

A total of three rounds of selection were performed on immobilized KDR,with varying protein concentrations and number of washings after theinitial binding process. After each round selection, 93 clones wererandomly picked and tested by phage ELISA for binding to KDR. Seventyout of the 93 clones (75%) picked after the second selection, andgreater than 90% of the recovered clones after the third selection werepositive in KDR binding, suggesting a high efficiency of the selectionprocess. DNA segments encoding the Fab from all the 70 bindersidentified in the second selection were amplified, digested with BstN I,and compared for fingerprint patterns. A total of 42 different patternswere observed, indicating an excellent diversity of the isolatedanti-KDR Fab. Cross-reactivity examination demonstrated that 19 out ofthe 42 antibodies were specific KDR-binders, whereas the rest 23antibodies bound to both KDR and its murine homologue, Flk-1. Furtherselection was achieved with a competitive VEGF-binding assay in whichthe binding of soluble KDR to immobilized VEGF in the presence orabsence of the anti-KDR Fab fragments was determined. The assayidentified four Fab clones that were capable of blocking the bindingbetween VEGF and KDR. Three were KDR-specific binders and onecross-reacted with Flk-1. DNA fingerprinting and sequencing analysisconfirmed that all four KDR/VEGF blocking antibodies were different(FIG. 1A) with unique DNA and amino acid sequences.

The amino acid sequences for CDR1, CDR2 and CDR3 of V_(H) and V_(L) forthe four clones are given in Table 1.

TABLE 1 CDR sequences of selected KDR-binding human Fabs Clone CDR1 CDR2CDR3 Light Chain D2C6 RASQSVSSYLA DSSNRAT LQHNTFPPT (SEQ ID NO:1) (SEQID NO:2) (SEQ ID NO:3) D2H2 RASQGISSRLA AASSLQT QQANRFPPT (SEQ ID NO:4)(SEQ ID NO:5) (SEQ ID NO:6) D1H4 AGTTTDLTYYDLVS DGNKRPS NSYVSSRFYV (SEQID NO:7) (SEQ ID NO:8) (SEQ ID NO:9) D1F7 SGSTSNIGTNTAN NNNQRPSAAWDDSLNGHWV (SEQ ID NO:10) (SEQ ID NO:11) (SEQ ID NO:12) Heavy ChainD2C6 GFTFSSYSMN SISSSSSYIYYADSVKG VTDAFDI (SEQ ID NO:13) (SEQ ID NO:14)(SEQ ID NO:15) D2H2 GFTFSSYSMN SISSSSSYIYYADSVKG VTDAFDI (SEQ ID NO:13)(SEQ ID NO:14) (SEQ ID NO:15) D1H4 GFTFSSYSMN SISSSSSYIYYADSVKG VTDAFDI(SEQ ID NO:13) (SEQ ID NO:14) (SEQ ID NO:15) D1F7 GGTFSSYAISGGIIPIFGTANYAQKFQG GYDYYDSSGVASPFDY (SEQ ID NO:16) (SEQ ID NO:17) (SEQID NO:18)Complete sequences for the V_(H) and V_(L) chains are presented in theSequence Listing. For D1F7, the nucleotide and amino acid sequences forV_(H) are represented by SEQ ID NOS:19 and 20 respectively, and thenucleotide and amino acid sequences for V_(L) are represented by SEQ IDNOS: 21 and 22.

For D2C6, the nucleotide and amino acid sequences for V_(H) arerepresented by SEQ ID NOS: 23 and 24 respectively, and the nucleotideand amino acid sequences for V_(L) are represented by SEQ ID NOS: 25 and26.

For D2H2, the nucleotide and amino acid sequences for V_(H) arerepresented by SEQ ID NOS: 30 and 31 respectively, and the nucleotideand amino acid sequences for V_(L) are represented by SEQ ID NOS: 32 and33.

For D1H4, the nucleotide and amino acid sequences for V_(H) arerepresented by SEQ ID NOS: 27 and 24 respectively, and the nucleotideand amino acid sequences for V_(L) are represented by SEQ ID NOS: 28 and29.

A second library was created combining the single heavy chain of D2C6with a diverse population of light chains derived from the originallibrary. Ten additional Fabs were identified, designated SA1, SA3, SB10,SB5, SC7, SD2, SD5, SF2, SF7, and 1121. The nucleotide and amino acidsequences for V_(L) of the ten Fabs are represented as follows. For SA1,the nucleotide and amino acid sequences for V_(L) are represented by SEQID NOS: 34 and 35. For SA3, the nucleotide and amino acid sequences forV_(L) are represented by SEQ ID NOS: 36 and 37. For SB10, the nucleotideand amino acid sequences for V_(L) are represented by SEQ ID NOS: 38 and39. For SB5, the nucleotide and amino acid sequences for V_(L) arerepresented by SEQ ID NOS: 40 and 41. For SC7, the nucleotide and aminoacid sequences for V_(L) are represented by SEQ ID NOS: 42 and 43. ForSD2, the nucleotide and amino acid sequences for V_(L) are representedby SEQ ID NOS: 44 and 45. For SD5, the nucleotide and amino acidsequences for V_(L) are represented by SEQ ID NOS: 46 and 47. For SF2,the nucleotide and amino acid sequences for V_(L) are represented by SEQID NOS: 48 and 49. For SF7, the nucleotide and amino acid sequences forV_(L) are represented by SEQ ID NOS: 50 and 51. For 1121, the nucleotideand amino acid sequences for V_(L) are represented by SEQ ID NOS: 52 and53.

The V_(L) CDR sequences are presented in Table 2.

TABLE 2 Light chain CDR sequences of KDR-binding human Fabs Clone CDR1CDR2 CDR3 SA1 TGSHSNFGAGTDV GDSNRPS QSYDYGLRGWV (SEQ ID NO:54) (SEQ ID(SEQ ID NO:56) NO:55) SA3 RASQNINNYLN AASTLQS QQYSRYPPT (SEQ ID NO:57)(SEQ ID (SEQ ID NO:59) NO:58) SB10 TGSSTDVGNYNYIS DVTSRPS NSYSATDTLV(SEQ ID NO:60) (SEQ ID (SEQ ID NO:62) NO:61) SB5 TGQSSNIGADYDVH GHNNRPSQSYDSSLSGLV (SEQ ID NO:63) (SEQ ID (SEQ ID NO:65) NO:64) SC7 RASQDISSWLAAASLLQS QQADSFPPT (SEQ ID NO:66) (SEQ ID (SEQ ID NO:68) NO:67) SD2RASQSIKRWLA AASTLQS QQANSFPPT (SEQ ID NO:69) (SEQ ID (SEQ ID NO:71)NO:70) SD5 SGSRSNIGAHYEVQ GDTNRPS QSYDTSLRGPV (SEQ ID NO:72) (SEQ ID(SEQ ID NO:74) NO:73) SF2 TGSSSNIGTGYDVH AYTNRPS QSFDDSLNGLV (SEQ IDNO:75) (SEQ ID (SEQ ID NO:77) NO:76) SF7 TGSHSNFGAGTDVH GDTHRPSQSYDYGLRGWV (SEQ ID NO:78) (SEQ ID (SEQ ID NO:80) NO:79) 1121RASQGIDNWLG DASNLDT QQAKAFPPT (SEQ ID NO:81) (SEQ ID (SEQ ID NO:83)NO:82)

Example II Assays Example II(a) Quantitative KDR Binding and Blocking ofKDR/VEGF Interaction

In a direct binding assay, various amounts of soluble Fab proteins wereadded to KDR-coated 96-well Maxi-sorp microtiter plates and incubated atRT for 1 h, after which the plates were washed 3 times with PBST. Theplates were then incubated at RT for 1 h with 100 μl of a rabbitanti-human Fab antibody-HRP conjugate (Jackson ImmunoResearch LaboratoryInc., West Grove, Pa.). The plates were washed and developed followingthe procedure described above for the phage ELISA. In a competitiveKDR/VEGF blocking assay, various amounts of Fab proteins were mixed witha fixed amount of KDR-AP (100 ng) and incubated at RT for 1 h. Themixtures were then transferred to 96-well microtiter plates precoatedwith VEGF₁₆₅ (200 ng/well) and incubated at RT for an additional 2 h,after which the plates were washed 5 times and the substrate for AP(p-nitrophenyl phosphate, Sigma) was added. Absorbance at 405 nm wasmeasured to quantify the bound KDR-AP molecules (8). IC₅₀, i.e., the Fabprotein concentration required for 50% inhibition of KDR binding toVEGF, was then calculated.

The four VEGF-blocking clones (D2C6, D2H2, D1H4, D1F7) were expressed assoluble Fab and purified from periplasmic extracts of E. coli by ProteinG affinity chromatography. The yield of purified Fab proteins of theseclones ranged from 60 to 400 μg/liter culture. SDS-PAGE analysis of eachpurified Fab preparation yielded a single protein band with expectedmolecular size (FIG. 1B).

FIG. 2 shows the dose-dependent binding of the anti-KDR Fab fragments toimmobilized receptor as assayed by a direct binding ELISA. Clone D2C6and D2H2 are more efficient binders, followed by clone D1H4 and D1F7.All four Fabs also block KDR binding to immobilized VEGF (FIG. 2B). Theantibody concentrations required for 50% of inhibition of KDR binding toVEGF are approximately 2 nM for clones D2C6, D2H2, and D1H4 and 20 nMfor clone DIF7. Only clone D1F7 blocks VEGF from binding to Flk-1 (FIG.2C), with an IC₅₀ of approximately 15 nM.

Example II(b) BIAcore Analysis of the Soluble scFv

The binding kinetics of soluble Fab proteins to KDR were measured bysurface plasmon resonance using a BIAcore biosensor (PharmaciaBiosensor). KDR-AP fusion protein was immobilized onto a sensor chip andsoluble Fab proteins were injected at concentrations ranging from 1.5 nMto 100 nM. Sensorgrams were obtained at each concentration and wereevaluated using a program, BIA Evaluation 2.0, to determine the rateconstants kon and koff. K_(d) was calculated from the ratio of rateconstants koff/kon.

All three KDR-specific Fab fragments bind to immobilized receptor withK_(d) of 2 to 4 nM (Table 3). The cross-reactive clone, D1F7, has aK_(d) of 45 nM, which is about 10- to 15-fold weaker than those of theKDR-specific clones. It is noteworthy that, although the overall K_(d)for the three KDR-specific Fab fragments are similar, the individualbinding kinetics, i.e., the kon and koff, for these antibodies are quitedifferent, e.g., D2C6 possesses the fastest on-rate, while D1H4 has theslowest off-rate (Table 3).

TABLE 3 Binding kinetics of the four neutralizing human anti-KDR Fabfragments Clone kon (10⁴ M⁻¹S⁻¹) koff (10⁻⁴ S⁻¹) Kd (nM) Hu-2C6 Fab 27.3± 8.6* 5.38 ± 0.54 1.97 Hu-2H2 Fab 12.4 ± 2.9  4.87 ± 0.18 3.93 Hu-1H4Fab 5.55 ± 0.59 1.53 ± 0.22 2.76 Hu-1F7 Fab 4.14 ± 1.21 18.7 ± 2.12 45.2*All numbers are determined by BIAcore analysis and represent the mean ±SE from at least three separate determinations.

Example 11(c) Binding Epitope Mapping

The production of KDR extracellular Ig-like domain deletion variants hasbeen previously described (Lu et al. (2000)). In an epitope-mappingassay, full length KDR-AP, Ap fusions of two KDR Ig-domain deletionvariants, and Flk-1-AP were first immobilized onto a 96-well plate(Nunc) using a rabbit anti-AP antibody (DAKO-immunoglobulins, Glostrup,Denmark) as the capture reagent. The plate was then incubated withvarious anti-KDR Fab proteins at RT for 1 h, followed by incubation witha rabbit anti-human Fab antibody-HRP conjugate. The plate was washed anddeveloped as described above.

The binding epitopes of the anti-KDR Fab fragments were mapped using thefull-length KDR and two KDR Ig domain-deletion variants. KDR (1-3) is aKDR variant containing the first three N-terminal Ig domains. KDR (3) isa variant containing only the third Ig domain. As shown in FIG. 3,clones D2C6 and D1H4 bind equally well to KDR, KDR (1-3) and KDR (3),thus locating their binding epitope(s) within Ig domain 3. Clones D2H2and D1F7 bind much more efficiently to full-length KDR and KDR (1-3),indicating a broader binding epitope(s) within KDR Ig domains 1 to 3.Only clone DIF7 cross-reacts with Flk-1.

Example II(d) Anti-Mitogenic Assay

HUVEC (5×10³ cells/well) were plated onto 96-well tissue culture plates(Wallach, Inc., Gaithersburg, Md.) in 200 μl of EBM-2 medium withoutVEGF, basic fibroblast growth factor (bFGF) or epidermal growth factor(EGF) and incubated at 37° C. for 72 h. Various amounts of Fab proteinswere added to duplicate wells and pre-incubated at 37° C. for 1 h, afterwhich VEGF₁₆₅ was added to a final concentration of 16 ng/ml. After 18 hof incubation, 0.25 μCi of [³H]TdR (Amersham) was added to each well andincubated for an additional 4 h. The cells were washed once with PBS,trypsinized and harvested onto a glass filter (Printed Filtermat A,Walach) with a cell harvester (Harvester 96, MACH III, TOMTEC, Orange,Conn.). The membrane was washed three times with H₂O and air-dried.Scintillation fluid was added and DNA incorporated radioactivity wasdetermined on a scintillation counter (Wallach, Model 1450 MicrobetaScintillation Counter).

The ability of human anti-KDR Fab to block VEGF-stimulated mitogenicactivity on HUVEC is shown in FIG. 4. All four human Fab fragmentsinhibited VEGF induced DNA synthesis in HUVEC in a dose-dependentmanner. The Fab concentration that inhibited 50% (EC₅₀) ofVEGF-stimulated [³H]-TdR incorporation in HUVEC, is approximately 0.5 nMfor clones D2C6 and D1H4, 0.8 nM for clone D2H2, and 15 nM for cloneD1F7. Controls included VEGF only (1500 cpm) and plain medium (60 cpm).Duplicate wells were assayed. The data shown are representative of atleast three separate experiments.

Example 11(e) Leukemia Migration Assay

HL60 and HEL cells were washed three times with serum-free plain RPMI1640 medium and suspended in the medium at 1×10⁶/ml. Aliquots of 100 μlcell suspension were added to either 3-μm-pore transwell inserts forHL60 cells, or 8-μm-pore transwell inserts for HEL cells (Costar®,Corning Incorporated, Corning, N.Y.) and incubated with the anti-KDR Fabproteins (5 μg/ml) for 30 min at 37° C. The inserts were then placedinto the wells of 24-well plates containing 0.5 ml of serum-free RPMI1640 with or without VEGF₁₆₅. The migration was carried out at 37° C.,5% CO₂ for 16-18 h for HL60 cells, or for 4 h for HEL cells. Migratedcells were collected from the lower compartments and counted with aCoulter counter (Model Z1, Coulter Electronics Ltd., Luton, England).

VEGF induced migration of HL60 and HEL cells in a dose-dependent mannerwith maximum stimulation achieved at 200 ng/ml (FIG. 5A). All theanti-KDR Fab fragments significantly inhibited VEGF-stimulated migrationof HL60 and HEL cells (FIG. 5B). As a control, a Fab fragment of C225,an antibody directed against EGF receptor, did not show significantinhibitory effect in this assay.

Example III Production of IgG Example 111(a) Construction of Vectors forExpression of IgG

Separate vectors for expression of IgG light chain and heavy chains wereconstructed. Cloned V_(L) genes were digested and ligated into thevector pKN100 (MRC. Cloned V_(H) genes were digested and ligated intothe vector pGID105 containing the human IgGI (γ) heavy chain constantdomain. pKN100 and pGID105 are available from the MRC, Constructs wereexamined by restriction enzyme digestion and verified bydideoxynucleotide sequencing. In both cases, expression is under controlof the HCMV promoter and terminated by an artificial terminationsequence.

The assembled heavy and light chain genes were then cloned into Lonza GSexpression vectors pEE6.1 and pEE12.1. Heavy and light chain vectorswere recombined into a single vectors for stable transfection of CHOcells and NS0 cells. Transfected cells were cultured in glutamine minusmedium and expressed antibodies at levels as high as Ig/L.

Example 111(b) Production and Characterization of Human Anti-KDR IgG

Both IMC-2C6 and IMC-1121 were produced in stably transfected NS0 celllines grown under serum-free conditions, and were purified from batchcell culture using Protein A affinity chromatography. The purity of theantibody preparations were analyzed by SDS-PAGE, and the concentrationswere determined by ELISA, using an anti-human Fc antibody as thecapturing agent and an anti-human κ chain antibody-horseradishperoxidase (HRP) conjugate as the detection agent. A clinical gradeantibody, IMC-C225, was used as the standard for calibration. Theendotoxin level of each antibody preparations was examined to ensure theproducts were free of endotxin contamination.

Anti-KDR antibodies were assessed for KDR binding and blocking of VEGFbinding. In the direct binding assay, various amounts of antibodies wereadded to KDR-coated 96-well Maxi-sorp microtiter plates (Nunc, Roskilde,Denmark) and incubated at room temperature (RT) for 1 h, after which theplates were washed 3 times with PBS containing 0.1% Tween-20. The plateswere then incubated at RT for 1 h with 100 μl of a rabbit anti-human IgGFc-HRP conjugate (Jackson ImmunoResearch Laboratory Inc., West Grove,Pa.). The plates were washed and developed as above. Human antibodiesIMC-2C6 and IMC-1121 were compared with IMC-1C11 (a mouse antibodyspecific for KDR) and IMC-C225 a chimeric antibody specific for EGFR).The anti-KDR antibodies bind to KDR in a dose-dependent manner, withIMC-1121 being the strongest binder (FIG. 6A).

The efficacy of the anti-KDR antibodies for blocking KDR from binding toVEGF was measured with a competition assay. Various amounts ofantibodies were mixed with a fixed amount of KDR-AP (100 ng) andincubated at RT for 1 h. The mixtures were then transferred to 96-wellmicrotiter plates precoated with VEGF₁₆₅ (200 ng/well) and incubated atRT for an additional 2 h, after which the plates were washed 5 times andthe substrate for AP (p-nitrophenyl phosphate, Sigma) was added,followed by reading the absorbance at 405 nm to quantify the boundKDR-AP molecules. IC₅₀, i.e., the antibody concentration required for50% inhibition of KDR binding to VEGF, was then calculated. The anti-KDRantibodies strongly blocked KDR from binding to VEGF (FIG. 6B), withsimilar potency. The IC₅₀ is approximately 0.8 to 1.0 nM for all threeantibodies. The control antibody, IMC-C225 (anti-human EGFR) does notbind KDR, and does not block KDR/VEGF interaction.

Antibody affinity or avidity was determined by BIAcore analysis, asabove. The binding kinetics, i.e., the association rate constant (kon)and the dissociation rate constant (koff), of the anti-KDR antibodieswere measured and the dissociation constant, K_(d), was calculated(Table 4).

TABLE 4 Binding kinetics of anti-KDR antibodies Antibody kon (10⁴ M⁻¹S⁻¹) koff (10⁻⁴ S⁻¹) Kd (nM) p1C11 scFv  7.7 ± 2.1*  1.0 ± 0.09 1.4 ±0.3 IMC-1C11 13.4 ± 2.9 0.37 ± 0.13 0.27 ± 0.06 Hu-2C6 Fab 17.1 ± 5.7 5.5 ± 0.76 3.6 ± 1.7 IMC-2C6 IgG 21.2 ± 8.1 0.43 ± 0.03 0.20 ± 0.01Hu-1121 Fab 29.6 ± 7.3 0.31 ± 0.06 0.11 ± 0.02 IMC-1121 IgG 47.9 ± 2.40.25 ± 0.04 0.05 ± 0.01 *All numbers are determined by BIAcore analysisand represent the mean ± SE from at least three separate determinations.IMC-1C11 binds to immobilized KDR with a dissociation constant (Kd) of0.27 nM, about 5-fold higher than its Fab counterpart. The Kd forIMC-2C6 is 0.2 nM, which is about 18-fold higher than that of themonovalent Hu-2C6 Fab, mainly due to an improvement in the off-rate.Affinity maturation of Hu-2C6 led to Hu-1121 Fab with a 33-foldimprovement in Kd (from 3.6 nM to 0.11 nM). Converting Hu-1121 Fab intobivalent IgG, IMC-1121, resulted in about 2-fold increase in overallbinding avidity.

Example 111(c) Inhibition of VEGF Binding to Cells and VEGF-StimulatedMitogenesis of HUVEC

In a cell-based radioimmunoassay, various amounts of anti-KDR antibodieswere mixed with a fixed amount (2 ng) of ¹²⁵I labeled VEGF₁₆₅ (R & DSystems) and added to a 80-90% confluent monolayer of HUVEC grown in a96-well microtiter plate. The plate was incubated at RT for 2 h, washed5 times with cold PBS, and the amounts of radioactivity that bound tothe endothelial cells were counted. As shown in FIG. 7A, anti-KDRantibodies competed efficiently with radiolabeled VEGF for binding toHUVEC. The data represent the means±SD for triplicate determinations.

The antibodies also blocked VEGF-stimulated HUVEC mitogenesis in adose-dependent manner (FIG. 7B). As described above for Fabs, variousamounts of the anti-KDR antibodies were first pre-incubated with growthfactor-starved HUVEC (5×10³ cells/well) at 37° C. for 1 h, after whichVEGF₁₆₅ was added to a final concentration of 16 ng/ml. After 18 h ofincubation, 0.25 μCi of [³H]-TdR (Amersham) was added to each well andincubated for an additional 4 h. The cells were washed, harvested, andDNA incorporated radioactivity was determined on a scintillationcounter. IMC-1121, the antibody with the highest affinity, is the mostefficacious inhibitor with an ED₅₀, i.e., the concentration that resultsin 50% of inhibition of [³H]-TdR incorporation, of about 0.7 nM, incomparison to that of 1.5 nM for both IMC-1C11 and IMC-2C6.

Example IV Inhibition of Leukemian Cells and Leukemia ProgressionExample IV(a) Expression of VEGF and KDR by Leukemia Cells

We examined VEGF and KDR expression, by RT-PCR, in three myeloid

leukemia cell lines: HL60 (promyelocytic); HEL (megakaryocytic); andU937 (histiocytic).

The following primers were used to amplify VEGF, Flt-1, KDR and theinternal control, α-actin: VEGF forward: 5′-TCGGGCCTCCGAAACCATGA-3′ (SEQID NO:86), and reverse: 5′-CCTGGTGAGAGATCTGGTTC-3′ (SEQ ID NO:87); Flt-1forward: 5′-TTTGTGATTTTGGCCTTGC-3′ (SEQ ID NO:88), and reverse:5′-CAGGCTCATGAACTTGAAAGC-3′ (SEQ ID NO:89); KDR forward:5′-GTGACCAACATGGAGTCGTG-3′ (SEQ ID NO:90), and reverse:5′-CCAGAGATTCCATGCCACTT-3′ (SEQ ID NO:91); α-actin forward:5′-TCATGTTTGAGACCTTCAA-3′ (SEQ ID NO:92), and reverse:5′-GTCTTTGCGGATGTCCACG-3′ (SEQ ID NO:93). The PCR products were analyzedon a 1% agarose gel. As shown in FIG. 8A, all three lines are positivefor VEGF expression, and HL60 and HEL, but not U937, are also positivefor KDR expression. The three cell lines are also positive for Flt-1expression as detected by RT-PCT (not shown).

VEGF production was examined for the three leukemia cell lines culturedunder either 10% FCS or serum-free conditions. The leukemia cells werecollected, washed with plain RPMI 1640 medium and seeded in 24-wellplates at density of 5×10⁵/ml, with or without the addition of 10% FCS.The cells were cultured at 37° C. for 72 hr, after which total numbersof cells were counted using a Coulter counter (Model Z1, CoulterElectronics Ltd., Luton, England) and the VEGF concentration in thesupernatant was determined using an ELISA kit (Biosource International,Camarillo, Calif.). The leukemia cells secrete significant amount ofVEGF when cultured in vitro (FIG. 8B), and both HL60 and U937 cellsproduced more VEGF under serum-starving conditions.

Example IV(b) Inhibition of VEGF-Induced Leukemia Cell Migration

Leukemia cell migration assays, as described in Example II(e), wereperformed with the three leukemia cell lines. The migration was carriedout for 16-18 h for HL60 cells, or for 4 h for HEL and U937 cells.

All three leukemia cell lines migrate in response to VEGF (FIG. 9).Incubation with anti-KDR antibodies inhibited, in a dose-dependentmanner, VEGF-induced migration of HL60 and HEL cells (FIGS. 9A and 9B),but had no effect on migration of U937 cells that does not express KDR(FIG. 9C). The VEGF-induced migration of U937 cells was, however,efficiently inhibited by an anti-human Flt-1 antibody, Mab 612 (FIG.9C). As expected, the anti-EGFR antibody, IMC-C225, showed no effect onVEGF-induced migration of human leukemia cells.

Example IV(b) Inhibition of Leukemia Growth In Vivo

6 to 8-week-old sex-matched (female) NOD-SCID mice were used in all theexperiments. The mice were irradiated with 3.5 Gy from a ¹³⁷Cs gamma-raysource at a dose rate of about 0.9 Gy/min and intravenously inoculatedwith 2×10⁷ HL60 cells. Three days after tumor inoculation, groups of 7to 9 mice were treated twice weekly with various doses of IMC-1C11,IMC-2C6 or IMC-1121 antibodies via intraperitoneal injection. Mice wereobserved daily for signs of toxicity and recorded for time of survival.For statistical analysis, the non-parametric one-tailed Mann-WhitneyRank Sum test was used.

All untreated mice died within 17 days (FIG. 10, mean time of survival,14±3 days). At this high tumor load, treatment with IMC-1C11 at 200μg/mouse/injection moderately increased the survival but all mice diedwithin 35 days (mean survival: 21±7 days; median survival 19 days,respectively. p=0.03 compared to the control group). IMC-2C6, given atthe same dose of 200 μg/mouse/injection, significantly prolonged themouse survival to 34±12 days (median=29 days. p<0.01 compared to thecontrol and p=0.01 compared to the INC-1C11-treated group). The antibodywith the highest affinity, IMC-1121, demonstrated a much strongeranti-leukemia effect, particularly with respect to IMC-1C11. The micetreated with IMC-1121 survived 63±12 days (median=60 days. p<0.001compared to both IMC-1C11 and IMC-2C6-treated groups). At a lowerantibody dose tested (100 μg/mouse/injection), IMC-1121 was also moreefficacious. Mice treated with the lower dose of IMC-1121 survived 46±16days (median=41 days). No overt toxicities were observed in any of theantibody-treated animals throughout the course of the experiment.

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

1.-29. (canceled)
 30. A method of neutralizing activation of KDR,inhibiting angiogenesis or reducing tumor growth, comprising contactinga cell with an effective amount of an isolated human antibody orfragment thereof which binds selectively to KDR, wherein said antibodycomprises complementarity determining regions represented by amino acidsequences selected from the group consisting of: (a) SEQ ID NO:1 atCDRL1, SEQ ID NO:2 at CDRL2, SEQ ID NO:3 at CDRL3, SEQ ID NO:13 atCDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3; (b) SEQ ID NO:4at CDRL1, SEQ ID NO:5 at CDRL2, SEQ ID NO:6 at CDRL3, SEQ ID NO:13 atCDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3; (c) SEQ ID NO:7at CDRL1, SEQ ID NO:8 at CDRL2, SEQ ID NO:9 at CDRL3, SEQ ID NO:13 atCDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3; (d) SEQ IDNO:10 at CDRL1, SEQ ID NO:11 at CDRL2, SEQ ID NO:12 at CDRL3, SEQ IDNO:16 at CDRH1, SEQ ID NO:17 at CDRH2, and SEQ ID NO:18 at CDRH3; (e)SEQ ID NO:54 at CDRL1, SEQ ID NO:55 at CDRL2, SEQ ID NO:56 at CDRL3, SEQID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3; (f)SEQ ID NO:57 at CDRL1, SEQ ID NO:58 at CDRL2, SEQ ID NO: 59 at CDRL3,SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3;(g) SEQ ID NO:60 at CDRL1, SEQ ID NO:61 at CDRL2, SEQ ID NO: 62 atCDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 atCDRH3; (h) SEQ ID NO:63 at CDRL1, SEQ ID NO:64 at CDRL2, SEQ ID NO:65 atCDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 atCDRH3; (i) SEQ ID NO:66 at CDRL1, SEQ ID NO:67 at CDRL2, SEQ ID NO:68 atCDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 atCDRH3; (j) SEQ. ID NO:69 at CDRL1, SEQ ID NO:70 at CDRL2, SEQ ID NO:71at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15at CDRH3; (k) SEQ ID NO:72 at CDRL1, SEQ ID NO:73 at CDRL2, SEQ ID NO:74at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15at CDRH3; (l) SEQ ID NO:75 at CDRL1, SEQ ID NO:76 at CDRL2, SEQ ID NO:77at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15at CDRH3; (m) SEQ ID NO:78 at CDRL1, SEQ ID NO:79 at CDRL2, SEQ ID NO:80at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15at CDRH3; and (n) SEQ ID NO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQ IDNO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ IDNO:15 at CDRH3.
 31. The method of claim 30, comprising neutralizingactivation of KDR.
 32. The method of claim 30, comprising inhibitingangiogenesis.
 33. The method of claim 30, comprising reducing tumorgrowth.
 34. The method of claim 30, wherein said antibody or fragmentthereof is selected from the group consisting of a single chainantibody, a Fab, a single chain Fv, a diabody, and a triabody.
 35. Themethod of claim 30, wherein said antibody or fragment thereof inhibitsbinding of VEGF to KDR.
 36. The method of claim 30, wherein saidantibody comprises complementarity determining regions represented bySEQ ID NO:1 at CDRLI, SEQ ID NO:2 at CDRL2, SEQ ID NO:3 at CDRL3, SEQ IDNO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3. 37.The method of claim 30, wherein said antibody comprises a light chainvariable domain represented by SEQ ID NO:26 and a heavy chain variabledomain represented by SEQ ID NO:24.
 38. The method of claim 30, whereinsaid antibody comprises complementarity determining regions representedby SEQ ID NO:81 at CDRLI, SEQ ID NO:82 at CDRL2, SEQ ID NO:83 at CDRL3,SEQ ID NO:13 at CDRHl, SEQ ID NO:14 at. CDRH2 and SEQ ID NO:15 at CDRH3.39. The method of claim 30, wherein said antibody comprises a lightchain variable domain represented by SEQ ID NO:53 and a heavy chainvariable domain represented by SEQ ID NO:24.
 40. The method of claim 30,wherein said antibody comprises a heavy chain variable domain selectedfrom the group consisting of SEQ ID NO:20, SEQ ID NO:24, and SEQ IDNO:31.
 41. The method of claim 30, wherein said antibody comprises alight chain variable domain selected from the group consisting of SEQ IDNO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53.
 42. The method ofclaim 30, comprising administering to a mammal an effective amount ofsaid antibody or fragment thereof.
 43. The method of claim 42, whereinsaid mammal is human.
 44. The method of claim 33, wherein said tumoroverexpresses KDR.
 45. The method of claim 33, wherein said tumor is anon-solid tumor.
 46. The method of claim 33, wherein said tumor isselected from the group consisting of a primary tumor, a metastatictumor and a refractory tumor.
 47. The method of claim 33, wherein saidtumor is selected from the group consisting of the brain, genitourinarytract, lymphatic system, stomach, colon, larynx, bone epidermoid,squamous, head and neck, pancreas, thyroid, liver and skin.
 48. Themethod of claim 33, wherein said tumor is selected from the groupconsisting of Kaposi's sarcoma, CNS neoplasm, neuroblastoma, capillaryhemangioblastoma, meningioma, cerebral metastases, leiomyosarcoma,squamous cell carcinoma, basal cell carcinoma, malignant keratinocytes.49. The method of claim 45, wherein said non-solid tumor is selectedfrom a group consisting of leukemia, multiple myeloma, and lymphoma. 50.The method of claim 33, wherein angiogenesis is involved invascularization and/or inflammation selected from the group consistingof atherosclerosis, rheumatoid arthritis (RA), neovascular glaucoma,proliferative diabetic retinopathy, macular degeneration, hemangiomas,angiofibromas, psoriasis, retrolental fibroplastic, corneal graftrejection, insulin-dependent diabetes mellitus, multiple sclerosis,myasthenia gravis, Chron's disease, autoimmune nephritis, primarybiliary cirrhosis, acute pancreatitis, allograph rejection, allergicinflammation, contact dermatitis, delayed hypersensitivity reactions,inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis,cognition defects induced by neuronal inflammation, Osler-Webersyndrome, restinosis, fungal infection, parasitic infection, viralinfection, and cytomegaloviral infection.
 51. The method of claim 33,wherein said tumor is a breast tumor.
 52. The method of claim 51,wherein said antibody comprises complementarity determining regionsrepresented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQ IDNO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ IDNO:15 at CDRH3.
 53. The method of claim 33, wherein said tumor is arenal tumor.
 54. The method of claim 53, wherein said antibody comprisescomplementarity determining regions represented by SEQ ID NO:81 atCDRL1, SEQ ID NO:82 at CDRL2, SEQ ID NO:83 at CDRL3, SEQ ID NO:13 atCDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3.
 55. The methodof claim 33, wherein said tumor is a melanoma tumor.
 56. The method ofclaim 55, wherein said antibody comprises complementarity determiningregions represented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQID NO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQID NO:15 at CDRH3.
 57. The method of claim 33, wherein said tumor is anovarian tumor.
 58. The method of claim 57, wherein said antibodycomprises complementarity determining regions represented by SEQ IDNO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQ ID NO:83 at CDRL3, SEQ IDNO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3. 59.The method of claim 33, wherein said tumor is a prostate tumor.
 60. Themethod of claim 59, wherein said antibody comprises complementaritydetermining regions represented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82at CDRL2, SEQ ID NO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 atCDRH2, and SEQ ID NO:15 at CDRH3.
 61. The method of claim 33, whereinsaid tumor is a lung tumor.
 62. The method of claim 61, wherein saidantibody comprises complementarity determining regions represented bySEQ ID NO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQ ID NO:83 at CDRL3, SEQID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3. 63.The method of claim 33, wherein said tumor is a colorectal tumor. 64.The method of claim 63, wherein said antibody comprises complementaritydetermining regions represented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82at CDRL2, SEQ ID NO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 atCDRH2, and SEQ ID NO:15 at CDRH3.
 65. The method of claim 33, whereinsaid tumor is a gastrointestinal tumor.
 66. The method of claim 65,wherein said antibody comprises complementarity determining regionsrepresented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82 at CDRL2, SEQ IDNO:83 at CDRL3, SEQ ID NO:13 at CDRH1, SEQ ID NO:14 at CDRH2, and SEQ IDNO:15 at CDRH3.
 67. The method of claim 33, wherein said tumor is asolid tumor.
 68. The method of claim 67, wherein said antibody comprisescomplementarity determining regions represented by SEQ ID NO:81 atCDRL1, SEQ ID NO:82 at CDRL2, SEQ ID NO:83 at CDRL3, SEQ ID NO:13 atCDRH1, SEQ ID NO:14 at CDRH2, and SEQ ID NO:15 at CDRH3.
 69. The methodof claim 33, wherein said tumor is a non-small cell lung tumor.
 70. Themethod of claim 69, wherein said antibody comprises complementaritydetermining regions represented by SEQ ID NO:81 at CDRL1, SEQ ID NO:82at CDRL2, SEQ ID NO:83 at CDRL3, SEQ ID NO:13 at CDRHl, SEQ ID NO:14 atCDRH2, and SEQ ID NO:15 at CDRH3.
 71. The method of claim 30, furthercomprising administering an antineoplastic agent.
 72. The method ofclaim 71, wherein said antineoplastic agent is a chemotherapeutic agentor radiation.
 73. The method of claim 30, further comprisingadministering a therapeutically effective amount of an epidermal growthfactor receptor antagonist.
 74. The method of claim 30, furthercomprising administering a therapeutically effective amount of fms-liketyrosine kinase receptor (flt-1) VEGFR-1.